Unlike simple retroviruses, lentiviruses encode a set of accessory proteins (Nef, Vif, Vpr, Vpu, and Vpx), each of which play a specific role in virus replication and pathogenesis, much of which are directed at evasion of the host adaptive or innate immune response (28, 33, 35, 36, 41, 46). Specifically, Nef downregulates cell surface CD4 molecules and disrupts antigen presentation, and Vif and Vpu serve to counteract the host restriction factors, APOBEC3G and BST-2/tetherin, respectively. Although the roles of Vpx and Vpr are less well understood, they are thought to aid in evasion of a yet uncharacterized restriction to virus replication in macrophages and dendritic cells (3, 7, 10, 18, 20, 22, 24, 27, 51). Sharing significant sequence and structural homology, the two proteins appear to have arisen from the duplication of a common ancestral precursor gene (44). Both are produced late in the viral life cycle and localize to the nucleus (5, 12, 31). A Vpr gene is present in the genome of all known lentiviruses, while Vpx is restricted to HIV-2 and the SIV of sooty mangabey (SIVsm) and macaque (SIVmac). Interestingly, SIV of the African green monkey encodes only Vpr, but this protein has some characteristics of Vpx, sharing the same virion packaging determinant and effect on virus replication in macrophages (1, 9).
A distinguishing feature of Vpr and Vpx is that they are packaged into virions at significant levels (2, 23, 50). Packaging of Vpr and Vpx occurs as the virion assembles and is mediated by amino acid motifs in the carboxy-terminal Gag protein p6, such that deletion of p6 prevents incorporation of both Vpr and Vpx (31, 38, 47). The packaging of Vpr and Vpx is also virus-specific (25). HIV-1 will not package SIVmac Vpr or Vpx, nor will SIVmac package HIV-1 Vpr. By analyzing viruses with mutated or truncated p6, various groups have identified amino acid motifs in p6 that mediate Vpr and Vpx incorporation. The motif 41LXXLF44 (SEQ ID NO: 58) near the p6 carboxy-terminus was reported by Kondo et al. as the major determinant required for the packaging of Vpr (30). Subsequently, Zhu et al. (53) reported that mutation of that motif did not prevent Vpr packaging. Instead, in a virus deleted for amino acids 35-52, the 15FRFG18 motif near the amino-terminus of p6 was required. This discrepancy has not yet been resolved. In SIVmac, the Vpx packaging motif has also been mapped to the N terminal region of p6, specifically to a conserved 17DXAXXLL23 (SEQ ID NO: 60) motif (1). Packaging of SIVagm Vpr was also dependent on this motif, drawing a resemblance with Vpx.
Vpr and Vpx are important for virus replication and pathogenicity in vivo, as demonstrated in the rhesus macaque model, where Vpx-deleted SIVmac239 is attenuated and Vpx/Vpr-deleted virus is further impaired (17). In vitro, neither Vpr nor Vpx is required for SIVmac replication in activated CD4 T cells (3, 18, 19). However, in monocyte-derived macrophages (MDM) and dendritic cells (MDDC), both proteins enhance SIVmac infection, although Vpr has a more modest effect. The presence of Vpx and Vpr in the virion suggests that they play a role early in virus replication. Vpr and Vpx were initially proposed to aid in nuclear import of the preintegration complex in infection of nondividing cells, a mechanism consistent with their karyophilic properties and their presence in the virion (5, 13). However, this view was challenged by the finding that HIV-1 deleted for Vpr maintained its ability to infect nondividing cells (48). Recently, a role for Vpx and Vpr in counteracting a restriction factor was suggested by the finding that both associated with an E3 ubiquitin ligase composed of damaged DNA binding protein 1 (DDB1), DDB1 cullen associated factor 1 (DCAF1), and Cullin 4A (Cul4A) (6, 21, 26, 39, 40, 42). By analogy with Vif, this finding suggested that Vpx and Vpr might act as substrate receptors that induce the ubiquitination of a host restriction factor (41, 43, 49). Evidence that Vpx plays a role in counteracting an MDM-specific host restriction was provided by somatic cell fusion experiments in which heterokaryons formed between MDM and COS were found to be nonpermissive for Vpx-deleted SIVmac (40). Furthermore, domain mapping of Vpx localized an activation domain at the amino-terminus that might serve as a binding site for the putative host restriction factor (21).
In studies investigating the mechanism by which Vpx promotes the infection of MDDC, Goujon et al. found that Vpx could facilitate infection when introduced into the target cell in trans (18, 20). For this, MDDC were exposed to virus-like particles (VLP) that contained Vpx and then were infected with Vpx-deleted SIVmac. The VLP dramatically enhanced the infectability of the cells. Additionally, Gramberg et al. found that VLP generated with a codon-optimized Vpx expression vector could boost the infection of MDM as much as 100-fold, while Vpr had little effect (21). In these cells, Vpx was found to relieve a block to infection at early reverse transcription or at uncoating. Interestingly, Vpx also dramatically enhanced HIV-1 infection of MDM and MDDC, even though the virus does not itself encode this accessory protein. Recently, Manel et al. used this property to achieve high levels of HIV-1 infected MDDC (32). The infected MDDC strongly induced innate immune defenses, resulting in the production of type-I IFN and upregulation of CD86. Surprisingly, the trigger that induced this response was not the in-coming virus but, rather, the newly produced Gag protein.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.